CN114805608B - A nanoparticle based on self-assembled ferritin anti-type A H1N1 subtype influenza virus and its preparation method and application - Google Patents

Abstract

The invention discloses a self-assembled ferritin-based anti-A/H1N1 subtype influenza virus nanoparticle, a preparation method and an application thereof, and relates to the technical field of genetic engineering. The nanoparticle is formed by connecting a signal peptide-ST-FE fusion protein and a signal peptide-SC-HA-VHH fusion protein in vitro; the signal peptide-ST-FE fusion protein is composed of a signal peptide-ST-FE fusion protein as shown in SEQ ID NO: 3 SpyTag is connected to the N-terminus of ferritin monomer subunit and obtains; Described signal peptide-SC-HA-VHH fusion protein is by the Nanobody N-terminus of anti-type A H1N1 subtype influenza virus hemagglutinin protein and as shown in SEQIDNO:4 The indicated signal peptide-SpyCatcher was ligated. The anti-H1N1 subtype influenza virus nanoparticle of the present invention has great significance for the monitoring, prevention and treatment of the A H1N1 subtype influenza virus.

Description

A self-assembled ferritin-based nanoparticle against H1N1 subtype influenza virus And its preparation method and application

technical field

The invention relates to the technical field of genetic engineering, in particular to a self-assembled ferritin-based anti-A/H1N1 subtype influenza virus nanoparticle, a preparation method and application thereof.

Background technique

Influenza is very common in actual production and life. It is caused by human or animal infection with influenza virus. The typical clinical symptoms of influenza include acute high fever, body pain, fatigue and respiratory symptoms. Influenza itself has obvious seasonality, high incidence in autumn and winter, some highly contagious viruses can cause large-scale or even global influenza pandemics, and there are very serious complications and even individual deaths due to differences in individual constitutions The phenomenon. Influenza viruses can be roughly divided into three different types, and these three different types are A, B, and C in turn, which can also be called A, B, and C.

H1N1 virus is a type of influenza A virus and one of the most commonly infected influenza viruses in humans. A number of studies have found that influenza A (H1N1) virus is a mutated new type of influenza A virus with four-source reassortment in its genome, which is a mixture of human influenza, swine influenza and avian influenza virus genes. The PB1 gene is derived from the human influenza H3N2 virus, the HA, NA, NP, NS and M genes are derived from the swine influenza H1N1 virus, and the PB2 and PA genes are derived from the avian influenza H1N1 virus. Human influenza is mainly caused by influenza A virus. Seasonal influenza can cause 250,000 to 500,000 deaths worldwide every year, and the number of deaths due to seasonal influenza in the United States can reach 3,000 to 49,000 per year. Influenza A virus has many subtypes, and most antibodies induced by different subtypes cannot provide cross-protection.

For a long time, genetically engineered antibodies have been one of the main means and research hotspots for the prevention and treatment of influenza A (H1N1) viruses. The development of a nanoparticle based on self-assembled ferritin against influenza A (H1N1) subtype virus has important practical significance for the prevention and treatment of influenza A (H1N1).

Contents of the invention

The purpose of the present invention is to provide a nanoparticle based on self-assembled ferritin anti-type A H1N1 subtype influenza virus and its preparation method and application, so as to solve the problems in the above-mentioned prior art. Combining the advantages of the nanobodies of the H1N1 subtype influenza virus to prepare nanoparticles against the A H1N1 subtype influenza virus, it is of great significance for the monitoring, prevention and treatment of the A H1N1 subtype influenza virus.

To achieve the above object, the present invention provides the following scheme:

The present invention provides a nanoparticle based on self-assembled ferritin against H1N1 subtype influenza virus, the nanoparticle is formed by linking signal peptide-ST-FE fusion protein and signal peptide-SC-HA-VHH fusion protein in vitro The signal peptide-ST-FE fusion protein is obtained by linking the signal peptide-SpyTag shown in SEQ ID NO:3 to the N-terminus of the ferritin monomer subunit; the signal peptide-SC-HA-VHH fusion protein It is obtained by linking the N-terminus of the nanobody against the hemagglutinin protein of the A/H1N1 subtype influenza virus to the signal peptide-SpyCatcher shown in SEQ ID NO:4.

Further, the amino acid sequence of the connecting peptide of the signal peptide-SpyTag and the N-terminal of the ferritin monomer subunit is shown in SEQ ID NO: 5; the nanobody against the hemagglutinin protein of the influenza A H1N1 subtype The amino acid sequence of the connecting peptide of N-terminal and signal peptide-SpyCatcher is shown in SEQ ID NO:5.

Further, the amino acid sequence of the signal peptide-ST-FE fusion protein is shown in SEQ ID NO:1; the amino acid sequence of the signal peptide-SC-HA-VHH fusion protein is shown in SEQ ID NO:2.

Further, the nucleotide sequence encoding the signal peptide-ST-FE fusion protein is shown in SEQ ID NO:10.

Further, the nucleotide sequence encoding the signal peptide-SC-HA-VHH fusion protein is shown in SEQ ID NO:11.

The present invention also provides a method for preparing the above self-assembled ferritin-based anti-A/H1N1 subtype influenza virus nanoparticles, comprising the following steps:

(1) Construct genes encoding the fusion proteins shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively, then transfer the constructed genes into vectors to construct expression plasmids, and then transfect to induce the expression of fusion proteins to obtain two fusion proteins protein;

(2) connecting the two fusion proteins obtained in step (1) in vitro to obtain the self-assembled ferritin-based anti-A/H1N1 subtype influenza virus nanoparticles.

Further, in step (2), the conditions for the in vitro connection are: 4°C, 16h.

The present invention also provides the application of the self-assembled ferritin-based anti-A/H1N1 subtype influenza virus nanoparticle in the preparation of a medicament for treating A/H1N1 subtype influenza.

The Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous linking peptide bond between Lys and Asp. This protein consists of two fragments: one is called SpyTag and the other is called SpyCatcher. The reaction is carried out under different pH, temperature and buffer conditions, and the reaction is simple and the yield is high. SpyTags can be fused terminally or internally and react specifically on the surface of mammalian cells. Peptide binding cannot be reversed by boiling or competing peptides.

There is a natural heavy chain antibody (Heavy chain antibody, HCAb) in camelids (dromedary, bactrian camel, llama, etc.) that only contains heavy chains and does not contain light chains. Heavy chain antibodies (HCAbs), which do not contain L-chain polypeptides, are unique in that they lack the first constant domain (CH1). In its N-terminal region, the H chain of the homodimeric protein contains a dedicated variable domain, termed VHH, for binding to its cognate antigen.

After cloning the variable region of the heavy chain antibody in camels, the obtained single-domain antibody consisting of only one heavy chain variable region is called variable domain of heavy chain of heavy-chain antibody (VHH), Its diameter is 2.5nm and its length is 4nm. It is known as the smallest unit that can bind target antigen. Its molecular mass is only 1/10 of that of monoclonal antibody. It is the smallest antibody unit with stable structure and antigen binding activity obtained so far. Therefore, Also known as nanobody (nanobody, Nb). As a small genetically engineered antibody, nanobodies have the advantages of high expression, water solubility, stability, tissue penetration and weak immunogenicity, which make the antibody have great potential in the fields of basic research and drug development. Broad application prospects. However, its single-targeting nanobody has the phenomenon of immune escape, so it is difficult to achieve the desired therapeutic effect.

Nanomaterials formed by self-assembled proteins not only have good biocompatibility, uniform particle size, and stability, but also have broad application prospects in cell imaging, lesion detection, and drug sustained release.

Ferritin is an important functional protein that participates in and regulates the storage and release of iron. It is a kind of protein with high iron content widely present in animal, plant and microbial cells. Naturally synthesized ferritin mostly presents a hollow spherical nano-cage structure with an outer diameter of 12nm and an inner diameter of 8nm. Trimeric subunits composed of protein subunits are assembled to form. When ferritin is used as a nanocarrier, it can wrap the target molecule inside the cage structure to realize the function of slow release or targeted release, and can also immobilize the target molecule on the outer surface of the cage structure to realize the functions of stabilizing the structure and exposing the target protein.

The target protein is linked to the N-terminus of the ferritin monomer subunit for fusion expression, so that the target antigen protein will be anchored on the outer surface of the self-assembled ferritin nanocage, and due to the unique assembly method of the ferritin subunit, it is suitable for The expression of antigens whose natural conformation is a trimer has a significant advantage. With the self-assembly of ferritin monomer subunits into trimers, the N-terminal fusion expressed antigens are very close in space and are easy to form a natural trimer structure. Such a trimer The body restores the natural conformation of the antigen protein to the greatest extent, and at the same time it is relatively stable, and the immunogenicity will be greatly enhanced compared with the expression alone. And due to its stability, ferritin can withstand high temperature and various denaturants without affecting its natural structure.

The invention discloses the following technical effects:

The first object of the present invention is to provide a method for realizing the multimerization of nanobodies, and another object of the present invention is to provide a nanoparticle that can directly and specifically bind to the influenza A (H1N1) subtype virus.

The present invention utilizes the characteristics of the spontaneous connection between SpyTag and SpyCatcher in the CnaB2 domain of Streptococcus pyogenes, and connects the signal peptide-SpyTag to the N-terminus of the ferritin monomer subunit for fusion expression. The N-terminus of the nanobody of the hemagglutinin protein is linked with the signal peptide-SpyCatcher for fusion expression. Finally, the two fusion proteins were connected in vitro, and the nanobody against the hemagglutinin protein of the A/H1N1 subtype influenza virus was displayed on the surface of the self-assembled ferritin cage structure, and finally the anti-A/H1N1 subtype influenza virus nanoparticles were realized preparation.

Compared with prokaryotic expression, the expression product expressed in the CHO eukaryotic cell system is appropriately modified and distributed regionally, and its immunogenicity is weak, and rejection is not easy to occur. In the process of protein expression, a signal peptide is introduced before the target protein, which can directly obtain the target protein in the cell culture medium, avoid cell disruption, improve protein purification efficiency and help improve protein purity, and further reduce non-target protein interference. Thereby improving the connection efficiency of SpyTag and SpyCatcher.

Compared with traditional nanobodies, the anti-H1N1 subtype influenza virus hemagglutinin protein nanoparticles have higher sensitivity and efficacy for the detection, diagnosis, prevention and treatment of H1N1 subtype influenza virus. Nanoparticles against influenza A (H1N1) subtype virus can improve the accuracy and repeatability of detection and diagnosis of influenza A (H1N1) subtype virus, which is of great significance for the monitoring of influenza A (H1N1) subtype virus.

The nanoparticle based on self-assembled ferritin anti-type A H1N1 subtype influenza virus prepared by the present invention is a multimerized nanobody, compared with type A H1N1 subtype influenza virus vaccine, the multimerized nanobody can directly treat the disease Treatment, avoiding the immune process, shortening the treatment process; compared with traditional nanobodies, multimerized nanobodies have higher sensitivity and curative effect on the detection, diagnosis, prevention and treatment of influenza A H1N1 subtype influenza virus, especially Nanoparticles against influenza A (H1N1) subtype virus can improve the accuracy and repeatability of detection and diagnosis of influenza A (H1N1) subtype virus, which is of great significance for the monitoring of influenza A (H1N1) subtype virus.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.

Figure 1 is a tandem structure diagram of gene signal peptide-ST-FE and signal peptide-SC-HA-VHH;

Fig. 2 is the agar gel electrophoresis figure of target protein gene;

Fig. 3 is the linearized agarose gel electrophoresis figure of plasmid signal peptide-ST-FE and signal peptide-SC-HA-VHH;

Fig. 4 is the western blot (Western Blot, WB) identification diagram of target protein gene;

Figure 5 is a signal peptide-ST-ferritin monomer subunit RT-PCR cell expression identification diagram;

Figure 6 is a WB identification diagram of the connection between ferritin monomer subunit and HA-VHH;

Figure 7 is electron microscope observation;

Figure 8 is a cell-level effect diagram of anti-influenza A H1N1 subtype influenza virus nanoparticles; the PBS group is no virus infection, PR8 is the influenza virus infection group, and antiviral particles+PR8 is the combined incubation group of antiviral particles and PR8 virus in vitro;

Figure 9 is a trend chart of body weight changes in mice;

Figure 10 is a trend chart of the survival rate of mice;

Fig. 11 is the ELISA figure of anti-H1N1 subtype influenza virus nanoparticle antigen recognition; Nbs-HA is the nanobody of traditional anti-HA, and F-Nbs-HA is the nanoparticle of anti-HA;

Figure 12 is a comparison chart of the antigen recognition ability between anti-H1N1 subtype influenza virus nanoparticles and traditional nanobodies; Nbs-HA is a traditional anti-HA nanobody, and F-Nbs-HA is an anti-HA nanoparticle.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail. The detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features and embodiments of the present invention.

It should be understood that the terminology described in the present invention is only used to describe specific embodiments, and is not used to limit the present invention. In addition, regarding the numerical ranges in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Any stated value or intervening value in a stated range, and each smaller range between any other stated value or intervening value in a stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents are described. In case of conflict with any incorporated document, the contents of this specification control.

It will be apparent to those skilled in the art that various modifications and changes can be made in the specific embodiments of the present invention described herein without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the present invention. The description and examples of the invention are illustrative only.

As used herein, "comprising", "comprising", "having", "comprising" and so on are all open terms, meaning including but not limited to.

The test methods used in the following examples are conventional methods unless otherwise specified; the experimental materials and reagents used are commercially available materials and reagents unless otherwise specified.

Example 1 Construction of plasmid signal peptide-SpyTag-ferritin monomer subunit (signal peptide-ST-FE) and signal peptide-SpyCatcher-anti-H1N1 subtype influenza virus hemagglutinin protein nanobody (signal peptide-SC-HA -VHH)

First, the gene signal peptide-ST-FE and signal peptide-SC-HA-VHH were synthesized. The gene tandem structure is shown in Figure 1, and the codons of the CHO eukaryotic expression system were used to optimize the codons, and were synthesized by GenScript Biotechnology Co., Ltd. The amino acid sequence (SEQ ID NO: 1) of the signal peptide-ST-FE is as follows:

MGWSCIILFLVATATGVHSAHIVMVDAYKPTKGGGSGGGSGGGSRMLKALNDQLNRELYSAYLYFAMAAYFEDLGLEGFANWMKAQAEEEIGHALRFYNYIYDRNGRVELDEIPKPPKEWESPLKAFEAAYEHEKFISKSIYELAALAEEEKDYSTRAFLEWFINEQVEEEASVKKILDK LKFAKDSPQILFMLDKELSARAPKLPGLLMQGGE;

The amino acid sequence (SEQ ID NO: 2) of the signal peptide-SC-HA-VHH is as follows:

MGWSCIILFLVATATGVHSSYYHHHHHHDYDIPTTENLYFQGSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGGSGGGSGGGSQVQLVESGGGLVQSGGSLRLSCAASGSMSRIITM GWYRQAPGMERELLAVIGNDNTVYGDSVQGRFTVSRDNAKNTAYLQMNSLNAEDTAMYYCKISTLTPPHEYWGQGTQVTVSSHHHHHH;

The amino acid sequence (SEQ ID NO: 3) of the signal peptide-SpyTag is as follows:

MGWSCIILFLVATATGVHSAHIVMVDAYKPTK;

The amino acid sequence (SEQ ID NO: 4) of the signal peptide-SpyCatcher is as follows:

MGWSCIILFLVATATGVHSSYYHHHHHHDYDIPTTENLYFQGSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI;

The amino acid sequence (SEQ ID NO: 5) of the connecting peptide is as follows:

GGGSGGGSGGGS;

The amino acid sequence (SEQ ID NO: 6) of the signal peptide is as follows:

MGWSCIILFLVATATGVHS.

After the signal peptide-ST-FE and signal peptide-SC-HA-VHH genes are synthesized, the synthetic gene is used as a template to amplify the target fragment by PCR technology. The amplification primers are:

Signal peptide-ST-FE:

Upstream primer sequence: AATCTCTAGAATGGGCTGGAGCTGCAT (SEQ ID NO: 7);

Downstream primer sequence: AATCAAGCTTTACTCGCCGCCCTGCAT (SEQ ID NO: 8);

Signal peptide-SC-HA-VHH

Upstream primer sequence: AATCTCTAGAATGGGCTGGAGCTGCAT;

Downstream primer sequence: AATCAAGCTTTTAGTGGTGGTGGTGGTG (SEQ ID NO: 9).

The nucleotide sequences of the signal peptide-ST-FE and signal peptide-SC-HA-VHH genes are as follows:

Signal peptide-ST-FE:

GAATTCATGGGTTGGAGTTGCATCATCCTATTTCTAGTGGCCACCGCTACCGGCGTGCACTCTGCCCACATCGTGATGGTGGACGCCTACAAGCCCACAAAGGGCGGAGGCAGCGGCGGCGGCTCTGGCGGAGGATCTCGGATGCTGAAGGCCCTGAACGACCAGCTGAATCGGGAGCTGTACTCCGCCTACCTGTACTTTGCCAT GGCCGCTTACTTCGAGGACCTGGGCCTGGAGGGCTTCGCCAACTGGATGAAAGCTCAGGCCGAGGAAGAGATCGGCCACGCCTTGAGATTCTACAACTACATCTACGACAGAAACGGCAGAGTGGAACTGGATGAGATTCCTAAGCCTCCAAAAGAGTGGGAGAGCCCCCTGAAGGCTTTCGAGGCTGCTTACGAGCATGAGAAGTTCATCT CCAAGTCCATCTACGAGCTGGCTGCTCTGGCAGAGGAAGAAAAGGATTATTCCACCAGAGCCTTCCTGGAATGGTTCATCAACGAGCAAGTCGAAGAAGAGGCCTCCGTGAAGAAGATCCTGGACAAGCTGAAGTTTGCCAAGGACTCCCCCTCAGATCCTGTTCATGCTCGATAAAGAACTGTCTGCTCGGGCCCCTAAGCTGCCTGGCCTGCTGATGCAGGGC GGCGAGTGAGGATCC (SEQ ID NO: 10);

Signal peptide-SC-HA-VHH:

GAATTCATGGGTTGGAGTTGCATCATCCTATTTCTAGTGGCCACCGCTACCGGCGTGCACTCCTCCTACTACCACCACCACCACCACCACGACTACGACATTCCCACCACCGAGAACCTGTACTTCCAGGGCTCCGCCACACACATCAAGTTTCCAAGAGAGACGAGGATGGCAAAGAGCTGGCTGGCGCTACAATGGAACTGAGAGATAGCTCTGGCAA AACAAATCTCTACCTGGATCAGCGACGGCCAAGTGAAGGACTTCTACCTCTATCCTGGCAAGTACACCTTCGTGGAAACAGCTGCTCCTGATGGCTACGAGGTGGCTACCGCCATCACCTTTACCGTGAACGAGCAGGGCCAGGTCACCGTGAACGGCAAGGCCACCAAGGGCGATGCCCACATCGGCGGAGGATCTGGCGGAGGCTCCGGCGGA GGCTCTCAGGTGCAGCTGGTGGAATCTGGAGGTGGCCTGGTGCAGTCCGGCGGCAGCCTGCGGCTGTCCTGTGCCGCTTCTGGCTCCATGAGCCGGATCATCACCATGGGCTGGTACAGACAAGGCCCCAGGCATGGAACGCGAGCTGGTCGCCGTGATCGGCAACAACGACAATACCGTTTACGGCGACTCCGTGCAAGGCAGATTCACCGT GTCTCGGGACAATGCCAAGAACACCGCTTATCTGCAGATGAACTCCCTGAACGCCGAGGACACCGCCATGTACTACTGCAAGATCTCCACCCTGACACCTCCTCACGAGTACTGGGGCCAGGGCACCCAGGTGACCGTGTCCTTCACCATCACCACCATCATTGAGGATCC (SEQ ID NO: 11).

The target gene amplification system is shown in Table 1:

Table 1

The PCR reaction program of the target gene is shown in Table 2:

Table 2

The amplified product was electrophoresed with 1% agarose gel, and the result was shown in Figure 2, which was the same as the expected fragment length.

The PCR product was recovered, and the two target fragments recovered by PCR gel were respectively digested with the eukaryotic expression plasmid P3 plasmid using restriction endonucleases Xba I and Hind III; the above two digested fragments were connected by T4 DNALigase to construct Two target gene carrier plasmids were obtained, ie, plasmid signal peptide-ST-FE (p3-ST-FE) and signal peptide-SC-HA-VHH (p3-SC-HA-VHH), and stored at -20°C.

Example 2 Linearization of Plasmid Signal Peptide-ST-FE and Signal Peptide-SC-HA-VHH

Take out the plasmid signal peptide-ST-FE and signal peptide-SC-HA-VHH stored at -20°C, and measure their concentrations with NanoDrop one to be 1200ng/μL and 890ng/μL, respectively, and use the restriction endonuclease PvuⅠ to perform single-enzyme Cut, the specific reaction system is shown in Table 3 and Table 4 below:

Table 3 Plasmid signal peptide-ST-FE single enzyme digestion reaction system

Table 4 Plasmid signal peptide-SC-HA-VHH single enzyme digestion reaction system

Add each component according to the single enzyme digestion reaction system table, and place in a 37°C water bath for enzyme digestion for 2 hours.

During digestion, prepare a 1% agarose gel. First prepare the gel plate, select and insert the comb according to the overall enzyme digestion system, then weigh 0.5g of agarose into a 100mL beaker, add 50mL of 1×TAE into it with a measuring cylinder, mix well and place in a microwave oven to heat Take it out for 30 seconds to observe the dissolution of the agarose, repeat the heating to make it fully dissolved, then add 2 μL of GoldView I nucleic acid dye to it, shake it gently (at this time, use even force to avoid bubbles), pour the gel, and let it stand for 40 minutes at room temperature to make the gel fully solidified. Then, mix the single-digestion product with an appropriate amount of 6×Loading Buffer and add them all to the gel well. The knockout plasmid without single-digestion and DL 10000 DNA Marker are used as controls. The electrophoresis conditions are constant voltage 110V, 600mA, 30min. After electrophoresis, the gel block was gently taken out and placed in a blue light gel cutter for gel cutting. According to the manual of SanPrepColumn DNA Gel Extraction Kit (SanPrepColumn DNA Gel Extraction Kit), the purified single-digested vector was recovered. The purified product was recovered, its concentration and OD _260/280 were measured using NanoDrop one, and stored at -20°C. 1% agarose gel as shown in Figure 3, the electrophoresis speed of the linearized plasmid is slower than that of the circular plasmid, as expected.

Example 3 Electrotransfection of Linear Signal Peptide-ST-FE and Signal Peptide-SC-HA-VHH to GS Gene-Deficient CHO Cells

(1) Preparation before electroporation of CHO suspension cells lacking GS gene. 24 hours before electroporation, the cells were inoculated in a 125 mL Erlenmeyer shaker flask with a viable cell density of 5×10 ⁵ cells/mL for shaking culture, the culture volume was 30 mL, and the speed of the carbon dioxide shaking incubator was set at 110 rpm.

(2) Collection of CHO suspension cells lacking GS gene. On the day of electroporation, take a sample to calculate the viable cell density and viability (98%) of the suspension culture, and calculate the volume of the cell suspension required to collect 1×10 ⁷ viable cells, place it in a sterile centrifuge tube, centrifuge at 1000rpm for 5min, and then re- Suspended in 400 μL of 302 medium (containing glutamine).

(3) Incubation of cells and plasmids. Add 40 μg of linearized knockout plasmid (linearized plasmid p3-ST-FE or linearized plasmid p3-SC-HA-VHH) to the above-mentioned treated cell suspension, gently pipette to mix, and incubate at room temperature for 10 minutes.

(4) electric shock. The parameters of the electrorotator are set in advance, and the electric shock conditions are: voltage 280V, electric shock time 20ms, electric shock once. After incubating at room temperature for 10 minutes, the above-mentioned cell plasmid mixture was quickly added to a pre-cooled 4mm electric shock cup, and electric shock was performed once.

(5) Cell treatment after electric shock. After the electric shock, the cells were quickly transferred to 20 mL of preheated 302 medium (containing glutamine), and the T75 cell culture flask was cultured for 24 hours.

(6) After 24 hours of electric shock, the cells were sampled to calculate the viability and recorded. After 24 hours of electroporation with linear plasmid p3-ST-FE, the cell viability rate was 48%, and after 24 hours of electroporation with linear plasmid p3-SC-HA-VHH, the cell viability rate was 52%.

(7) Continue culturing with a glutamine-free medium until the cell viability exceeds 95%.

Example 4 Screening of Cell Lines After Plasmid Signal Peptide-ST-F and Plasmid Signal Peptide-SC-HA-VHH Electroporation

After 24 hours of electroporation, the static culture cell pool is a mixed group of stably transfected cells, which contains the cell line that stably expresses the target protein we need. Since the cells contain the GS gene after the plasmid electroporation is successful, the medium without glutamine is used. Screening, screening out monoclonal cell lines by the method of limiting dilution, the specific steps are as follows:

(1) Preparation of conditioned medium for subcloning plating. 24 hours before subcloning plating, the suspension cells after electroporation were inoculated at 1×10 ⁶ cells/mL in a 125 mL Erlenmeyer shaker flask for shaking culture at a rotation speed of 110 rpm and a culture volume of 30 mL.

(2) On the day of subcloning plating, place the cells inoculated and cultured in step 1 in a 50 mL sterile centrifuge tube, centrifuge at 1000 rpm for 5 min, and use a 0.2 μm sterile disposable filter to filter out the cell debris in the cell culture supernatant as a condition The culture medium is ready for use.

(3) Plating medium configuration method: 60% fresh 302 medium (without glutamine) + 30% conditioned medium + 10% fetal bovine serum (FBS) + penicillin and streptomycin (PS), mix the above ingredients Preheat at 37°C for later use.

(4) Plank. After 24 hours of electroporation, the cells were sampled to calculate the living cell density and viability according to the steps of cell counting. Use a 96-well cell culture plate to screen monoclonal cell lines. Considering that monoclonal cells may die during the growth process, serially dilute the electroporated cells in the plating medium for 24 hours to a final density of 1 cell per 100 μL, and the diluted cells The suspension was added to a 96-well cell culture plate at 100 μL/well with a multichannel pipette gun, and five 96-well cell culture plates were evenly spread, and placed in a 37°C, 5% CO ₂ incubator for culture.

(5) On the 7th day after subcloning was plated, the growth of the clones was observed under a microscope. The monoclonals resembled circular cell clusters radiating outward from the central point, and the single and polyclonal wells were marked at the same time. Replace the medium in the monoclonal well once every 5-7 days as needed.

(6) Select cell lines with good growth status for future use.

The western blot (Western Blot, WB) identification of embodiment 5 target protein

The selected and cultured cell lines were inoculated at 5×10 ⁵ cells/mL in a 125 mL Erlenmeyer shaker flask for shaking culture, the culture volume was 30 mL, and the speed of the carbon dioxide shaking incubator was set at 110 rpm. The viability of the cell lines was measured every 12 hours. When the cell viability reached 70%, the cell supernatant was taken for WB identification of the target protein, as shown in Figure 4. Conforms to expected stripe size. Signal peptide-ST-FE without HIS tag has no band, and signal peptide-SC-HA-VHH with signal peptide shows a band.

(1) Glue compounding: the lower layer of glue is 10% separating glue. The upper gel is 5% stacking gel

Wash the comb and let it dry naturally. Brush the glass plate clean and drain the water against the glue stand. (Ultrapure water rinse)

Check for leaks with ultrapure water. Then mix the gel according to the recipe. When adding 10% SDS, pour out the leak detection water, blot dry with filter paper, then continue to add the remaining 10% APS and TEMED and mix well.

Add the prepared separation gel along one part of the glass, about 4.5mL per piece, seal it with ultrapure water immediately after adding the glue (to make the surface of the glue flat), and wait for an obvious refraction between the separation gel and the water On the surface, start to configure according to the formula of the stacking gel. When adding 10% SDS, pour off the water sealed, dry it with filter paper, and then continue to add the remaining 10% APS and TEMED and mix well.

Pour the prepared stacking gel into the plate, quickly insert the comb, wait for the upper layer of gel to be completely solidified, then pull out the comb, place the gel together with the glass plate in the electrophoresis tank for electrophoresis, or place it in the electrophoresis buffer, and place it in the 4 degrees refrigerator, set aside.

(2) Spotting

Mix the sample with an appropriate amount of loadingbuffer and boil for 5min, then add 10-15μL to each empty space. Blank wells were replaced with 1×SDS buffer.

(3) run electrophoresis

Set to voltage 100V. Generally, electrophoresis takes 90-120 minutes, and the specific situation is determined according to the progress of electrophoresis.

During the running of electrophoresis, it is necessary to prepare the things for the membrane transfer and configure the membrane transfer buffer (stored at 4°C and used for later use).

Cutting the film: According to the number of samples, cut the film a little longer to the length of a hole.

Bubble film: First soak the film with methanol for more than ten seconds, rinse it with ultra-pure water (just rinse it once), soak it in the transfer buffer, and put it in the refrigerator for later use.

Soak the filter paper, put the filter paper in a tray, soak it with the transfer buffer washing solution, and put it in the refrigerator for later use. Take the ice when there are more than ten minutes before the film transfer.

(4) transfer film

Put the transfer tank into a box with ice, take it out from the -20°C refrigerator and put it into the tank, pour the transfer buffer into the tank, take out all the transfer supplies that were originally placed in the refrigerator, The order of placement when transferring membranes: Because the protein is electrophoresed from the negative electrode to the positive electrode, it is placed separately from the positive electrode to the negative electrode: sponge-two layers of filter paper-PVDF membrane-glue-two layers of filter paper-sponge (to prevent the gap between the gel and the filter paper, the glue and the There are air bubbles between the membrane, between the glue and the filter paper).

Stack several layers and tighten them into the slot. Set the current to 300mA, set the button to the time gear, and transfer the film for 120 minutes. There are more than ten minutes before the end of the membrane transfer, and 5% BSA blocking solution (blocking solution: dissolve 2g of BSA in 40mL TBST)

(5) closed

Take out the film and turn it over, soak it in 5% BSA, and shake it in the refrigerator at 4 degrees for 1 hour. Or seal at room temperature for 1h (according to the room temperature at that time)

(6) primary antibody

The primary antibody diluent was 1% BSA, and an appropriate volume of HIS primary antibody (mouse source) was taken and diluted at a ratio of 1:800-1000. Incubate overnight in a shaker at 4°C.

(7) washing

Wash with TBST three times, each time for 5 minutes, during which the secondary antibody was prepared, and the secondary antibody was rabbit anti-mouse antibody.

(8) Secondary antibody: Incubate on a shaker at room temperature for 1 h in the dark.

(9) Washing: wash three times with TBST, 5 min each time.

(10) EXPOSURE.

The RT-PCR detection of embodiment 6 signal peptide-ST-FE

Since the signal peptide-ST-FE protein does not contain a HIS tag, its RNA was extracted to detect whether it was transcribed. The result is shown in Figure 5, in line with the expected size.

(1) The cells (10 ⁷ cells) were centrifuged to discard the culture medium, and washed twice with PBS. Discard PBS and blot.

(2) Add 1mL TOIZOL (Total RNA Extraction Reagent) for grinding.

(3) Add 250 μm chloroform, shake vigorously for 30 seconds, and let stand on ice for 12 minutes.

(4) Centrifuge at 4°C and 13500rpm/min for 15min.

(5) Mix 400 μL of the supernatant with an equal amount of isopropanol and a new EP tube, and freeze at 4°C for 35 minutes. Then centrifuge at 4°C, 13500rpm/min for 10min.

(6) Discard the supernatant, add 1 mL of 75% ethanol, and shake gently. Centrifuge at 4°C, 10600rpm/min for 5min.

(7) Discard the supernatant, let stand or blot dry with filter paper. Add 10 μL deionized water to dissolve for later use or store at -80°C.

(8) Reverse transcribe the extracted RNA according to Table 5, and perform electrophoresis on 1% agarose gel. The result is shown in FIG. 5 , which is the same as the expected fragment length.

table 5

The purification of embodiment 7 target protein

ST-FE protein was purified using size exclusion HPLC. SC-HA-VHH protein was purified by HIS protein purification column, and imidazole was removed by 3KD dialysis membrane. Finally, 0.5 mg/ml of ST-FE protein and 0.45 mg/ml of SC-HA-VHH protein were obtained.

Example 8 In vitro connection of target protein and electron microscope observation

The ST-FE monomer protein and the SC-HA-VHH protein were mixed in equimolar amounts, and connected continuously for 16 hours at 4°C. Obtain anti-A/H1N1 subtype influenza virus nanoparticles. According to the characteristics of the HIS tag, use WB to detect the connection between the two. As shown in Figure 6, the connection is displayed, which is in line with the expected size. The ST-FE protein and the FE-HA-VHH linker were observed using transmission electron microscopy, respectively. As shown in Figure 7, under the same magnification, the diameter of FE-HA-VHH linker particles is significantly larger than that of FE protein, which is in line with expectations.

Example 9 Antiviral Identification of Anti-Influenza A H1N1 Subtype Influenza Virus Nanoparticles at the Cell Level

Before the experiment, prepare 1×10 ⁶ MDCK cells and spread them on 6 empty plates. The obtained anti-influenza A H1N1 subtype virus nanoparticles and influenza A H1N1 subtype influenza virus (PR8) were mixed in equal amounts to form a 0.5 mL mixed solution and incubated at 37° C. for 15 min, and the virus amount was 10 ³ PFU. And observe the result after 72h, as shown in Figure 8. The results show that the anti-H1N1 subtype influenza virus nanoparticles can effectively bind to the A/H1N1 subtype influenza virus (PR8) and inhibit the infection of the A/H1N1 subtype influenza virus (PR8) on MDCK cells.

Example 10 In vivo antiviral identification of anti-H1N1 subtype influenza virus nanoparticles

6-8 weeks old Balb/c female SPF mice were divided into 2 groups, 8 mice in each group. One group is the PBS group, and the other is the anti-A/H1N1 subtype influenza virus nanoparticle treatment group. 4 hours before infection with influenza A (H1N1) subtype influenza virus (PR8), each mouse in the PBS group was instilled with 200 μL of PBS in the nasal cavity, and in the anti-influenza A (H1N1) subtype influenza virus nanoparticle treatment group, each mouse was instilled with 200 μL of anti-influenza A (H1N1) in the nasal cavity. Subtype influenza virus nanoparticles (50 μg), the amount of virus infected with each mouse was 2×LD50, 24 hours after virus infection, each mouse in the PBS group was instilled with 200 μL PBS in the nasal cavity, and the anti-H1N1 subtype influenza virus nanoparticle treatment group Each mouse was intranasally instilled with 200 μL anti-A/H1N1 subtype influenza virus nanoparticles (50 μg). The body weight and survival rate of the mice were recorded. The experimental results are shown in Figures 9 and 10, which show the changes in body weight and survival rate of the mice. The mice in the anti-H1N1 subtype influenza virus nanoparticle treatment group had no significant changes in body weight, no abnormal performance, and no death. The mice in the PBS control group had obvious clinical symptoms, such as rough coat, shortness of breath, hunchback, slow movement, clustering, and significant weight loss. All mice died within 8 days.

Example 11 Detection of Anti-Influenza A H1N1 Subtype Influenza Virus Nanoparticles and Virus Binding Ability

Firstly, the binding ability of anti-influenza A H1N1 subtype influenza virus nanoparticles to influenza A H1N1 subtype influenza virus (PR8) was tested. Dilute H1N1 subtype influenza virus (PR8) to 10 ⁶ PFU, add 50 μL to each well of the ELISA plate, place at 37°C for 2 hours, or 4°C for 24 hours; discard the liquid in the well (to avoid evaporation, the plate should be Cover or place the plate flat in a metal wet box with wet gauze on the bottom). Seal the enzyme-labeled reaction wells: add 2% BSA to 200 μL/well, block at 37°C for 60 minutes or at 4°C for 24 hours; remove the air bubbles in each well, and wash the wells with washing solution three times after sealing, each time for 3 minutes. Washing method: blot the reaction solution in the well, fill the plate well with the washing solution, add the washing solution again after aspiration and discard and repeat 3 times. Pour off liquid and pat dry on absorbent paper. The number of washes is 3 times. Anti-A/H1N1 subtype influenza virus nanoparticles were 50 μL per well, and 3 gradients were set, and placed at 37°C for 40-60min. Wash the wells with washing solution 3 times, 3 min each time. Add HIS primary antibody overnight at 4°C, and wash as before. Add the enzyme-labeled antibody (secondary antibody) according to the reference working dilution provided by the enzyme conjugate provider. 37 ℃, between 30-60min, add 50μL to each well. Wash the same as before. Add substrate solution (prepared for current use), TMB-hydrogen peroxide urea solution is the first choice, followed by OPD-hydrogen peroxide substrate solution system. Substrate addition amount: 50 μL per well, place at 37°C in the dark for 3-5 minutes, add stop solution to develop color. Stop the reaction: Add 50 μL of stop solution to each well to stop the reaction, and measure the experimental results within 20 minutes. Judgment of results: OPD uses 492nm wavelength after color development, and 450nm wavelength is required for TMB reaction product detection. At the same time, a positive group and a negative group were set up, with 3 repetitions in each group. Traditional nanobody treatment is the same as above. The results show that the anti-influenza A H1N1 subtype virus nanoparticles can be combined with the A H1N1 subtype influenza virus (PR8), and the sensitivity is better than that of traditional nanobodies. The results are shown in Figure 11.

Then set 6 gradients for H1N1 subtype influenza virus (PR8), which are 10 ⁶ PFU, 5×10 ⁵ PFU, 2×10 ⁵ PFU, 1×10 ⁵ PFU, 5×10 ⁴ PFU, 10 ³ PFU And PBS is set as a control, and an equal amount of anti-A/H1N1 subtype influenza virus nanoparticles is combined, and the steps are the same as above. Traditional nanobody treatment is the same as above. The results showed that the maximum specific binding of anti-H1N1 subtype influenza virus nanoparticles was better than that of traditional nanobodies, and the binding sensitivity to AH1N1 subtype influenza virus nanoparticles was 7.18 times higher than that of traditional nanobodies. The results are shown in Figure 12.

The above-mentioned embodiments are only to describe the preferred mode of the present invention, and are not intended to limit the scope of the present invention. Variations and improvements should fall within the scope of protection defined by the claims of the present invention.

sequence listing

<110> Qingdao Agricultural University

<120> A nanoparticle based on self-assembled ferritin against H1N1 subtype influenza virus and its preparation method and application

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Thr Val Asn Glu Gln Gly Gln Val Thr Val Asn Gly Lys Ala Thr Lys

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Gly Asp Ala His Ile Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly

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Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ser Gly

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Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Met Ser Arg Ile

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Ile Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Met Glu Arg Glu Leu

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Val Ala Val Ile Gly Asn Asn Asp Asn Thr Val Tyr Gly Asp Ser Val

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Gln Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Thr Ala Tyr

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Leu Gln Met Asn Ser Leu Asn Ala Glu Asp Thr Ala Met Tyr Tyr Cys

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Lys Ile Ser Thr Leu Thr Pro Pro His Glu Tyr Trp Gly Gln Gly Thr

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Gln Val Thr Val Ser Ser His His His His His His His His

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aacgacaata ccgtttacgg cgactccgtg caaggcagat tcaccgtgtc tcgggacaat 660

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tactgcaaga tctccaccct gacacctcct cacgagtact ggggccagggg cacccaggtg 780

accgtgtcct ctcaccatca ccaccatcat tgaggatcc 819

Claims (6)

1. A nanoparticle based on self-assembled ferritin anti-type A H1N1 subtype influenza virus, characterized in that, the nanoparticle is composed of signal peptide-ST-FE fusion protein and signal peptide-SC-HA-VHH fusion protein in vitro connected; the signal peptide-ST-FE fusion protein is obtained by connecting the signal peptide-SpyTag shown in SEQ ID NO: 3 to the N-terminus of the ferritin monomer subunit; the signal peptide-SC-HA- The VHH fusion protein is obtained by linking the N-terminus of the nanobody against the hemagglutinin protein of the influenza A H1N1 subtype virus with the signal peptide-SpyCatcher shown in SEQ ID NO: 4; The amino acid sequence of the connecting peptide of the N-terminal of the signal peptide-SpyTag and the ferritin monomer subunit is shown in SEQ ID NO: 5; the nanobody N-terminus and The amino acid sequence of the connecting peptide of the signal peptide-SpyCatcher is shown in SEQ ID NO: 5; The amino acid sequence of the signal peptide-ST-FE fusion protein is shown in SEQ ID NO:1; the amino acid sequence of the signal peptide-SC-HA-VHH fusion protein is shown in SEQ ID NO:2. 2. the nanoparticle based on self-assembled ferritin anti-type A H1N1 subtype influenza virus according to claim 1, is characterized in that, the nucleotide sequence of coding described signal peptide-ST-FE fusion protein is as SEQ ID NO : 10. 3. the nanoparticle based on self-assembled ferritin anti-type A H1N1 subtype influenza virus according to claim 1, is characterized in that, the nucleotide sequence of coding described signal peptide-SC-HA-VHH fusion protein is as SEQ ID NO: 11. 4. a method for preparing the nanoparticle based on self-assembled ferritin anti-type A H1N1 subtype influenza virus as claimed in claim 1, is characterized in that, comprises the following steps: (1) Construct genes encoding the fusion proteins shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively, then transfer the constructed genes into vectors to construct expression plasmids, and then transfect to induce the expression of fusion proteins to obtain two fusion proteins protein; (2) connecting the two fusion proteins obtained in step (1) in vitro to obtain the self-assembled ferritin-based anti-A/H1N1 subtype influenza virus nanoparticles. 5. The preparation method according to claim 4, characterized in that, in step (2), the conditions for the in vitro connection are: 4°C, 16h. 6. A use of the nanoparticle according to any one of claims 1-3 in the preparation of a medicament for treating influenza A (H1N1) subtype.

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